Cell growth and division are controlled by the temporal expression of different sets of genes, allowing the dividing cell to progress through the different phases of the cell cycle. Continued growth and organogesis in plants requires precise function of the cell cycle machinery. Plant development, which is directly affected by cell division rates and patterns, also is influenced by environmental factors, such as temperature, nutrient availability, light, etc. See Gastal and Nelon, Plant Physiol. 105:191-7 (1994), Ben-Haj-Sahal and Tardieu, Plant Physiol. 109:861-7 (1995), and Sacks et al., Plant Physiol. 114:519-27 (1997). Plant development and phenotype are connected with the cell cycle, and altering expression of the genes involved in the cell cycle can be a useful method of modifying plant development and altering plant phenotype.
The ability to alter expression of cell cycle genes is extremely powerful because the cell cycle drives plant development, including growth rates, responses to environmental cues, and resulting plant phenotype. Control of the plant cell cycle and phenotypes associated with alteration of cell cycle gene expression, in the vascular cambium, in particular, has applications for, inter alia, alteration of wood properties and, in particular, lumber and wood pulp properties. For example, improvements to wood pulp that can be effected by altering cell cycle gene expression include increased or decreased lignin and cellulose content, and altered length, diameter, and lumen diameter of cells. Manipulating the plant cell cycle, and in particular the cambium cell cycle (i.e. the rate and angle of cell division), can also engineer better lumber having increased dimensional stability, increased tensile strength, increased shear strength, increased compression strength, increased shock resistance, increased stiffness, increased or decreased hardness, decreased spirality, decreased shrinkage, and desirable characteristics with respect to weight, density, and specific gravity.
A. Cell Cycle Genes and Proteins
1. Cyclin Dependent Protein Kinase
Progression through the cell cycle is regulated primarily by cyclin-dependent kinases (CDKs). CDKs are a conserved family of eukaryotic serine/threonine protein kinases, which require heterodimer formation with a cyclin subunit for activity. For review see, e.g. Joubes et al., Plant Mol. Biol. 43: 607-20 (2000), Stals and Inze, Trends Plant Sci. 6:359-64 (2001), and John et al., Protoplasma 216: 119-42 (2001).
The are five subclasses of CDK's, each having a different cyclin binding consensus sequence. In CDK type A the cyclin binding consensus sequence is PSTAIRE (SEQ ID NO: 778). Id. The cyclin binding consensus sequence in CDK types B-1, B-2, and C are PPTTLRE (SEQ ID NO: 779), PPTALRE (SEQ ID NO: 780), and PITAIRE (SEQ ID NO: 781), respectively. Joubes et al, Plant Physiol, 126: 1403-15 (2001).
Cell cycle progression is directed, in part, by changes in CDK activity. CDK activity is modulated by a number of different cell cycle protein components, such as changes in the abundance of individual cyclins due to changing rates of biosynthesis and proteolysis. Fluctuations in cyclin concentrations result in commensurate fluctuations in CDK activity. Cyclin accumulation is especially important in terminating the G1 phase of the cell cycle because DNA replication is initiated by an increase in CDK activity.
Activation of CDK also requires phosphorylation of a threonine residue within the T-loop of CDK by a CDK-activating kinase (CAK). Umeda et al., Proc. Nat'l Acad. Sci. U.S.A. 97: 13396-400 (2000). It was suggested by Yamaguchi et al., Plant J. 24: 11-20 (2000), that cyclin H is a regulatory subunit of CAK. CDK activity is further regulated by interaction with a CDK regulatory subunit, a small (70-100 AA) protein involved in cell cycle regulation.
A cell must exit the cell cycle in order to commit to differentiation, senescence or apoptosis. This process involves the down-regulation of CDK activities. CDK inhibitors (CKI) are low molecular weight proteins, which are important for cell cycle regulation and development. CKIs bind stoichiometrically to CDK and down-regulate the activity of CDKs.
Many biochemical properties of ICK1, the first plant CKI to be identified from Arabidopsis thaliana, are known. Wang et al., Nature 386:451-2 (1997) Wang et al., Plant J. 24: 613-23 (2000). ICK1 is expressed at low levels in many tissue types, and there can be a threshold level of ICK1 that must be overcome before a cell can enter the cell cycle. Wang et al., Plant J. 24: 613-23 (2000). ICK1 is induced by the plant growth regulator abscisic acid (ABA), which inhibits cell division by blocking DNA replication. When the expression of ICK 1 increases, there is a corresponding decrease in Cdc2-like H1 histone activity. ICK1 has been shown to bind in vitro with the cyclins C2c2a and CycD3, and deletion experiments have identified different domain regions for these two interactions.
Altering the expression of CDK regulatory protein or a subunit thereof is known to cause changes in plant phenotype. Overexpression of the Arabidopsis CDK regulatory subunit, CKS1At, resulted in a reduction of leaf size, root growth rates and meristem size. Additionally, overexpression of CKS1At resulted in inhibition of cell-cycle progression, with an extension in the duration of the G1 and G2 phases of the cell cycle.
2. Cyclins
Cyclins are positive regulatory subunits of cyclin-dependent kinase (CDK) enzymes and are required for CDK activity. Fowler et al., Mol. Biotech. 10, 123, 126. Cyclins and CDK complexes provide temporal regulation of transition through the cell cycle. Evidence also suggests that cyclins provide spatial regulation of specific CDK activity, differentially targeting the cytoskeleton, spindle, phragmoplast, nuclear envelope, and chromosomes.
Plant cyclins are classified into five major groups: A, B, C, D, and H. Renaudin et al., Plant Mol. Biol. 32: 1003-18 (1996) and Yamaguchi et al., (supra 2000). Cyclins can be divided into mitotic cyclins (A and B) and G1 cyclins.
The mitotic cyclins possess a consensus sequence (R-x-x-L-x-x-I-x-N. SEQ ID NO: 782) located at the N-terminal region, termed a destruction box, adjacent to a lysine-rich region. The destruction box and lysine-rich region target the mitotic cyclins for ubiquitin-dependent proteolysis during mitosis. Stals, supra at 361, and Fowler, supra at 126. The destruction box in A versus B cyclins differs slightly and this difference is thought to result in slightly different timing of degradation of A versus B cyclins. Fowler, supra at 126. A-type cyclins accumulate during the S, G2, and early M phase of the cell cycle, whereas B-type cyclins accumulate during the late G2 and early M phase. Mironov et al., Plant Cell 11: 509-22 (1999). Three subgroups of A-type cyclins are known in plants, but only one is known in animals. Cyclin A1 (cycA1;zm;1 from Zea cans) is most concentrated during cytokinesis at the microtubule-containing phragmoplast. Expression of cyclin A2 is upregulated by auxins in roots, and by cytokinins in the shoot apex. Abrahams et al., Biochim. Biophys. Acta 28: 1-2 (2001).
D-type cyclins, of which five subgroups are known, are thought to control the progression through the G1 phase in response to growth factors and nutrients. Riou-Khamlichi et al., Mol. Cell Biol. 20: 4513-21 (2000). For example, the expression of D-type cyclins is upregulated by sucrose as shown by an increase in cycD2 mRNA 30 minutes after sucrose exposure, and an increase in cycD3 four hours after sucrose exposure. This timing corresponds to early G1-phase and late G1-phase, respectively. Cockcroft et al., Nature 405: 575-9 (2000). Furthermore, in Arabidopsis, a D3 cyclin was shown to be upregulated by the brassinosteroid, epi-brassinolide.
Cyclin D2 proteins bind with CDKA to produce an active complex, which binds to and phosphorylates retinoblastoma-related protein (Rb). This process is found in actively proliferating tissue, suggesting it plays an important function during late G1- and early S-phase. Three different D3-type cyclins are active during tomato fruit development. These proteins all contain a retinoblastoma binding motif and a PEST-destruction motif. There are differences in the spatial and temporal expression of these D3 cyclins, inferring different roles during fruit development.
Overexpression of cyclin D was shown to increase overall growth rate. Over-expression of cyclin D2 in tobacco increases causes shortening the G1-phase which producing a faster rate of cell cycling.
C- and H-type cyclins were characterized in poplar (Populus tremula ×tremuloides) and rice (Oryza sativa) but their exact function is still unclear. Putative cyclins with a lesser degree of peptide sequence conservation have also been identified. For example, Arabidopsis CycJ18 has only 20% identity with homologues over the cyclin box domain. CycJ18 is expressed predominantly in young seedlings. Arabidopsis F3O9.13 protein also has similarity to the cyclin family.
3. Histone Acetyltransferase/Deacetyltransferase
Histone acetyltransferase (HA) and histone deacetyltransferase (HAD) control the net level of acetylation of histones. Histone acetylation and deacetylation are thought to exert their regulatory effects on gene expression by altering the accessibility of nucleosomal DNA to DNA-binding transcriptional activators, other chromatin-modifying enzymes or multi-subunit chromatin remodeling complexes capable of displacing nucleosomes. Lusser et al., Nucleic Acids Res. 27: 4427-35 (1999). Therefore, in general, the HDAs are involved in the repression of gene expression, while HAs are correlated with gene activation.
HA effects acetylation at the ε-amino group of conserved lysine residues clustered near the amino terminus of core histones which up-regulates gene expression.
HDAs remove acetyl groups from the core histones of the nucleosome. There are numerous family members in the HDA group, many of which are conserved throughout evolution. Lechner et al., Biochim Biophys Acta 5:181-8 (1996). HDAs fimction as part of multi-protein complexes facilitating chromatin condensation.
HDAs and HAs recognize highly distinct acetylation patterns on the nucleosome. It is thought that different types of HDAs interact with specific regions of the genome, to influence gene silencing.
Schultz et al., Genes Dev. 15: 428-43 (2001), demonstrated that the superfamily of Kruppel-associated-box zinc finger proteins (KRAB-ZFPs) are linked to the nucleosome remodelling and histone deacetylation complex via the PHD (plant homeodomain) and bromodomains of co-repressor KAP-1, to form a cooperative unit that is required for transcriptional repression. A maize HDAC (HD2) has been identified that has no sequence homology to other eukaryotic HDACs, but instead contains sequence similarity to peptidyl-prolyl cis-trans isomerases (PPIases).
The effects of interfering with histone deacetylation are discussed in e.g. Tian and Chen, Proc. Nat'l Acad. Sci. USA 98: 200-5 (2001).
4. Peptidyl Prolyl Cis-Trans Isomerase
Peptidylprolyl isomerases (e.g., peptidylprolyl cis-trans isomerase, peptidyl-prolyl cis-trans isomerase, PPIase, rotamase, cyclophilin) catalyze the interconversion of peptide bonds between the cis and trans conformations at proline residues. Sheldon and Venis, Biochem J. 315: 965-70 (1996). This interconversion is thought to be the rate limiting step of protein folding. PPIases belong to a conserved family of proteins that are present in animals, fungi, bacteria and plants. PPIases are implicated in a number of responses including the response to environmental stress, calcium signals, transcriptional repression, cell cycle control, etc. Viaud, et al., Plant Cell 14: 917-30 (2002).
5. Retinoblastoma-Related Protein
Retinoblastoma (Rb)-related protein putatively regulates progression of the cell cycle through the G1 phase and into S phase. Xie et al., EMBO J. 15: 4900-8 (1996) and Ach et al., Mol. Cell Biol. 17: 5077-86 (1997).
Although Rb is well-characterized in mammalian systems, the role of Rb-related proteins in regulation of G1 phase progression and S phase entry is not well characterized in plants. It is known, however, that RB-related protein functions through its association with various other cellular proteins involved in cell cycle regulation, such as the cyclins, WD40 proteins, Soni et al., Plant. Cell. 7:85-103 (1995); Grafi et al., Proc. Natl. Acad. Sci. U.S.A. 93:8962 (1996); Ach et al., Plant Cell 9:1595-606 (1997); Umen and Goodenough, Genes Dev. 15:1652-61 (2001); Mariconti et al., J. Biol. Chem. 277:9911-9 (2002).
6. WD40 Repeat Protein
WD40 is a common repeating motif involved in many different protein-protein interactions. The WD40 domain is found in proteins having a wide variety of functions including adaptor/regulatory modules in signal transduction, pre-mRNA processing and cytoskeleton assembly. Goh et al., Eur. J. Biochem. 267: 434-49 (2000).
The WD40 domain, which is 40 residues long, typically contains a GH dipeptide 11-24 residues from the N-terminus and the WD dipeptide at the C-terminus. Id. Between the GH dipeptide and the WD dipeptide lies a conserved core which serves as a stable platform where proteins can bind either stably or reversibly. The core forms a propeller-like structure with several blades. Each blade is composed of a four-stranded anti-parallel β-sheet. Each WD40 sequence repeat forms the first three strands of one blade and the last strand in the next blade. The last C-terminal WD40 repeat completes the blade structure of the first WD40 repeat to create the closed ring propeller-structure. The residues on the top and bottom surface of the propeller are proposed to coordinate interactions with other proteins and/or small ligands.
Studies in yeast demonstrated that Cdc20, which contains the WD40 motif, is required for the proteolysis of mitotic cyclins. This process is mediated by an ubiquitin-protein ligase called anaphase-promoting complex (APC) or cyclosome. Following ubiquitination and proteolysis by the 26S proteasome, the cell can segregate chromosomes, and exit from mitosis. Cdc20 also contains a destruction-box domain.
7. WEE1-Like Protein
WEE1 controls the activity of cyclin-dependent kinases. WEE1 itself is a serine/threonine kinase. Sorrell et al., Planta 215: 518-22 (2002). The enzymatic activity of these protein kinases is controlled by phosphorylation of specific residues in the activation segment of the catalytic domain, sometimes combined with reversible conformational changes in the C-terminal autoregulatory tail. This process is conserved among eukaryotes, from fungi to animals and plants. Similarly, there is a high degree of homology between WEE1 proteins from various organisms. For example, there is 50% identity between the protein kinase domains of the human and maize WEE1 proteins.
Expression of WEE1 is shown to occur only in actively dividing tissues and is believed to inhibit cell division by acting as a negative regulator of mitosis. WEE1 is believed to prevent entry from G2 to M by protecting the nucleus from cytoplasmically-activated cyclin B1-complexed CDC2 before the onset of mitosis. For example, over-expression of AtWEE1 (from Arabidopsis) and ZmWEE1 (from Zea cans) in fission yeast inhibits cell division which results in elongated cells. Sun et al., Proc. Nat'l Acad. Sci. USA 96: 4180-5 (1999).
B. Expression Profiling and Microarray Analysis in Plant Development
The multigenic control of plant phenotype presents difficulties in determining the genes responsible for phenotypic determination. One major obstacle to identifying genes and gene expression differences that contribute to phenotype in plants is the difficulty with which the expression of more than a handful of genes can be studied concurrently. Another difficulty in identifying and understanding gene expression and the interrelationship of the genes that contribute to plant phenotype is the high degree of sensitivity to environmental factors that plants demonstrate.
There have been recent advances using genome-wide expression profiling. In particular, the use of DNA microarrays has been useful to examine the expression of a large number of genes in a single experiment. Several studies of plant gene responses to developmental and environmental stimuli have been conducted using expression profiling. For example, microarray analysis was employed to study gene expression during fruit ripening in strawberry, Aharoni et al., Plant Physiol. 129:1019-1031 (2002), wound response in Arabodopsis, Cheong et al., Plant Physiol. 129:661-7 (2002), pathogen response in Arabodopsis, Schenk et al., Proc. Nat'l Acad. Sci. 97:11655-60 (2000), and auxin response in soybean, Thibaud-Nissen et al., Plant Physiol. 132:118. Whetten et al., Plant Mol. Biol. 47:275-91 (2001) discloses expression profiling of cell wall biosynthetic genes in Pinus taeda L. using cDNA probes. Whetten et al. examined genes which were differentially expressed between differentiating juvenile and mature secondary xylem. Additionally, to determine the effect of certain environmental stimuli on gene expression, gene expression in compression wood was compared to normal wood. 156 of the 2300 elements examined showed differential expression. Whetten, supra at 285. Comparison of juvenile wood to mature wood showed 188 elements as differentially expressed. Id. at 286.
Although expression profiling and, in particular, DNA microarrays provide a convenient tool for genome-wide expression analysis, their use has been limited to organisms for which the complete genome sequence or a large cDNA collection is available. See Hertzberg et al., Proc. Nat'l Acad. Sci. 98:14732-7 (2001a), Hertzberg et al., Plant J, 25:585 (2001b). For example, Whetten, supra, states, “A more complete analysis of this interesting question awaits the completion of a larger set of both pine and poplar ESTs.” Whetten et al. at 286. Furthermore, microarrays comprising cDNA or EST probes may not be able to distinguish genes of the same family because of sequence similarities among the genes. That is, cDNAs or ESTs, when used as microarray probes, may bind to more than one gene of the same family.
Methods of manipulating gene expression to yield a plant with a more desirable phenotype would be facilitated by a better understanding of cell cycle gene expression in various types of plant tissue, at different stages of plant development, and upon stimulation by different environmental cues. The ability to control plant architecture and agronomically important traits would be improved by a better understanding of how cell cycle gene expression effects formation of plant tissues, how cell cycle gene expression causes plant cells to enter or exit cell division, and how plant growth and the cell cycle are connected. Among the large number of genes, the expression of which can change during development of a plant, only a fraction are likely to effect phenotypic changes during any given stage of the plant development.